The ability to clone PCR products is of universal importance in the field of molecular biology. There are several general texts which address the subject, including Sambrook et. al., Molecular Cloning--A Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989 and PCR Protocols A Guide to Methods and Applications (Innis et. al. eds) Academic Press Inc. San Diego, Calif. (1990) which are incorporated herein by reference.
The T. aquaticus DNA polymerase 1 (Taq) has an activity which frustrated many people who attempted to clone PCR products before its nature was recognized. Taq has a weak terminal transferase activity under standard PCR conditions which typically causes the addition of a single adenosine (A) residue onto the 3' end of the amplified DNA product. Using traditional cloning strategies this residue must either be polished off using an exonuclease, or severed as a result of an internal restriction site cleavage by a restriction endonuclease prior to cloning. The problems with either of these strategies (detailed below) are familiar to those skilled in the art.
Polishing in preparation for blunt-ended cloning is generally straightforward, but blunt-ended cloning is often difficult, requiring relatively high concentrations of target and vector DNA. In addition, when a particular orientation of the cloned PCR DNA product relative to the vector is desired there must be an additional step of selecting for the desired orientation in the cloning protocol. Moreover, the cloning vector must either possess a blunt restriction site, or must itself be polished in order to ligate the PCR product into the vector. This requirement may necessitate additional cloning steps in order to create the desired final DNA construct.
Restriction enzyme digestion of PCR products for cloning typically requires that there be a known internal restriction site. This is usually accomplished by incorporating restriction sites into the PCR primers, a strategy with four distinct disadvantages. First, if the sequence of the PCR amplicon is unknown, then one may inadvertently select a restriction site in designing the primer which is also present in the target amplicon, leading to the cloning of unwanted DNA fragments. Second, restriction enzymes typically require a target with a minimum amount of surrounding DNA in order to recognize the restriction site in the proper context for cleavage to occur; thus, in designing a primer it is necessary to incorporate DNA residues between the restriction site and the end of the DNA molecule. The number of residues which are necessary for establishing the proper context is specific to each restriction enzyme and not always known; moreover, the addition of extra bases beyond the restriction site can be costly, particularly when it is done on a routine basis. Third, in some restriction enzymes the recognition site and the cleavage site are separated (e.g., HphI), which generally makes it impossible to use them in the cloning strategy. Finally, the addition of restriction sequences and the necessary contextual DNA for endonuclease cleavage reduces the specificity of the primer for the amplicon, a problem which is especially acute when attempting to use degenerate primers in the PCR amplification.
More recently, persons of skill have taken advantage of the terminal transferase activity of taq by designing cloning vectors to utilize the overhanging 3' A residue found on PCR DNA products (Hernstadt et. al., international patent application number PCT/US 91/07147, International Publication Number WO 92/06/06189 (4-16-92)). While this strategy is certainly useful, it is limited to a subset of all possible vectors, requiring many investigators to re-clone the cloned PCR product to create a desired construct.
The current methodology substantially overcomes each of the above enumerated problems. In general, an alkane diol residue such as 1,3-propanediol is incorporated into the PCR primer during oligonucleotide synthesis, which acts as a block to DNA chain elongation by taq during PCR. By strategically selecting the 5' ends of the PCR primers during oligonucleotide synthesis it is possible to generate a defined 5' overhang on all PCR products, without the need for further treatment. After ligating the PCR products into the vector of choice and transforming a bacterial cell such as E. coli, the non-basic residue is excised from the transformation vector by the cell's endogenous DNA repair machinery.
The generation of cDNA from mRNA is a commonly practiced technique in the field of molecular biology. It is often necessary to create a clone of a gene which lacks introns--particularly when the gene is to be expressed in a prokaryotic cell, which in general do not process their RNA to splice out introns. If a gene's product is of commercial interest it is often easiest given the current state of the art to express the gene in a bacterial cell culture and recover the protein in bulk.
cDNA libraries are often used in the process of isolating a gene of interest, or in order to help define the structure of a gene. cDNA libraries are superior to genomic libraries for a number of applications, including the creation of expression libraries and the creation of enriched libraries (libraries created from tissue in which the gene product is thought to be expressed). Due to the general utility of cDNA libraries, considerable effort and creativity has gone into methods which improve the heterogeneity of the library, which clone the cDNA constructs into expression vectors and which generate more nearly full-length clones.
All available methods for cDNA cloning suffer from common limitations. The creation of specific ends on the cDNA for cloning is problematic. Blunt ended cloning artificially enriches a library for the frequently found cDNAs in a sample due to the low rate of ligation, making it inappropriate for the generation of rare cDNAs. Adding adaptors to the cDNA molecule by incorporating restriction sites into the primers used in the first round of synthesis suffers from many of the same problems as the analogous strategy described above for PCR. Cloning the tailed cDNA molecule requires that the incorporated restriction site be cleaved by the appropriate restriction endonuclease, which may result in truncated cDNAs where there is an internal endonuclease site in the cDNA molecule. In addition, if the cleavage reaction is inefficient (i.e., if the endonuclease cleaves a site near the end of the molecule with lower than usual efficiency), artificial enrichment for frequently found cDNA molecules will occur.
The present invention overcomes the enumerated limitations of the prior art by incorporating an abasic residue into the first-round cDNA primer which blocks chain elongation by the DNA polymerase used in the cDNA synthesis at the site on the opposing strand opposite the abasic residue, creating a 5' overhang compatible with a cleaved vector. A strategy for generating a 5' overhang at the opposite end of the cDNA molecule using a primer complementary to a homopolymeric tail, in which the primer has an abasic residue is also described. Once the cDNA is ligated into the vector of choice, the construct is transformed into a bacterial cell via standard methods and the abasic residues are excised by the bacterial cell's endogenous repair machinery.